Among the insect-borne flaviviruses, the four dengue virus serotypes are most important in terms of human morbidity and geographic distribution. Several live-attenuated dengue vaccine formulations are currently in clinical trials. However, the safety of such dengue vaccines remains a concern. Passive immunization using monoclonal antibodies from humans or non-human primates represents an attractive alternative to a dengue vaccine. Repertoire cloning was performed to recover Fab antibody fragments from bone marrow mRNA of a chimpanzee infected with all four dengue virus serotypes. We have identified several Fab antibodies highly efficient for neutralization of dengue type 4 virus from a panel of Fabs recovered by repertoire cloning following panning with dengue type 4 virus. One of the neutralizing Fabs was selected to construct a full-length IgG1 antibody in combination with the human sequences. The humanized antibody produced in mammalian CHO cells proved to be highly neutralizing for dengue type 4 virus in vitro. In order to recover Fab antibodies against the other three dengue virus serotypes, repertoire cloning of the same phage library was also performed using dengue type 1, 2 and 3 viruses as panning antigens. Instead of a type-specific antibody, dengue virus-neutralizing Fabs that were cross-reactive with all dengue serotypes were identified. One of these Fabs also neutralized the West Nile virus among other flaviviruses tested. The humanized antibody derived from this Fab proved to be efficient for cross-neutralization of dengue type 1 and type 2 viruses. This antibody also cross-neutralized dengue type 3 and type 4 viruses and West Nile virus, but at a slightly reduced titer. Both the dengue type 4-specific and the cross-neutralizing, humanized monoclonal antibodies represent attractive candidates for further development of a passive immunization strategy against dengue and perhaps other flaviviruses-associated diseases. To gain an insight into the serological specificity and functional activity, we have also determined the epitope determinants of the chimpanzee Fab antibody, which had been shown to be broadly reactive to flaviviruses and efficient for neutralization of dengue type 1 and type 2 viruses. Analysis of dengue type 2 antigenic variants showed that a determinant critically involved in the Fab antibody binding and neutralization mapped to glycine at position 106 within the flavivirus-conserved fusion loop in the envelope glycoprotein. Another determinant affecting antibody neutralization, but not antibody binding, mapped to histidine at position 317 that is spatially closed to glycine-106 of the opposite homodimer of the dengue type 2 virus envelope protein. From the locations of these amino acids in the three-dimensional structure, the antibody appears to recognize a novel epitope on the envelope glycoprotein that has not been mapped before.